Screening artificial nucleic acids by particle display

ABSTRACT

The invention provides xeno-nucleic acid particle display libraries, methods for identifying functional non-natural nucleic acid (XNA) aptamers using the particle display libraries, and compositions comprising XNA aptamers identified by screening candidate molecules using the xeno-nucleic acid particle display libraries.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/842,022, filed May 2, 2019 which is hereby incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION

Aptamers, nucleic acid molecules that mimic antibodies by folding intoshapes with ligand binding affinity (Ellington and Szortak, 1990, Nature346:818-822), have enormous potential as diagnostic and therapeuticagents (Cho et al., 2009, Annu Rev Anal Chem 2:241-264; Zhou and Rossi,2017, Nat. Rev. Drug Discov. 16:181-202). Aptamers are generated by invitro selection or SELEX (systematic evolution of ligands by exponentialenrichment) from large libraries of combinatorial sequences (Wilson andSzortak, 1999, Ann. Rev. Biochem. 68:611-647). Similar to naturalselection, in vitro selection is a process of selective amplification inwhich a population of nucleic acid molecules is challenged to bind adesired target or catalyze a chemical reactions. Molecules having adesired fitness are recovered and amplified to generate a new populationof molecules that has become enriched in members with a particularactivity. Although hundreds of aptamers have been reported in theliterature, the vast majority of these sequences are unsuitable for invivo applications because they are susceptible to digestion by enzymesthat degrade DNA and RNA (Keefe et al., 2010, Nat. Rev. Drug Discov.9:537-550). Even aptamers with modified bases or expanded geneticletters, which have shown tremendous promise in array-based diagnosticsor have achieved high target binding affinity, respectively, are proneto nuclease attack (Gold et al., 2010, Plos One, 5: e15004; Kimoto etal., 2013, Nat. Biotechnol. 31:453-457). One exception is Spiegelmers,mirror-image aptamers composed of L-DNA or L-RNA, but such reagents arecurrently restricted to achiral targets or targets that can be generatedby chemical synthesis, which is a very small fraction (<1%) of the humanproteome (Vater and Klussmann, 2015, Drug Discov Today, 20:47-155).

Thus, there is a need in the art for novel methods for synthesis ofbiologically stable aptamers. The present invention satisfies this unmetneed by establishing an XNA aptamer particle display format for rapidlyscreening aptamers composed of artificial genetic polymers (also knownas xeno-nucleic acids or XNAs) for high affinity and high specificity toa desired target.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a method of producing amonoclonal xeno-nucleic acid aptamer particle (mXNAP) display librarycomprising: (a) providing a population of particles comprising aplurality of clonal template DNA molecules, each of which comprises aDNA coding region and a fixed sequence primer binding site; (b) ligatinga self-priming stem-loop forming hairpin DNA molecule to the 3′ end ofthe template DNA molecules; (c) extending the 3′ end of the self-primingstem-loop in the presence of a polymerase capable of synthesizing an XNAfrom a DNA template and one or more XNA triphosphate molecules (xNTPs)to form a population of particles comprising clonal double strandedXNA-DNA hybrid molecules; (d) contacting the double stranded XNA-DNAdisplay templates with a primer which anneals to the loop region of thestem-loop structure; and (e) extending the DNA primer using dNTPs and aDNA polymerase to displace the XNA portion of the XNA-DNA displaytemplates to form a population of mXNAP display particles comprising aplurality of clonal nucleic acid molecules comprising a dsDNA region anda single-stranded XNA region.

In one embodiment, the mXNAP library is a mTNAP, mHNAP, mCeNAP, mLNAP,mANAP, mphNAP, or mFANAP library.

In one embodiment, the polymerase capable of synthesizing an XNA from aDNA template and one or more XNA triphosphate molecules (xNTPs) isKod-RSGA, Kod-RS, Kod-RI, Therminator, pol6G12_521L, pol6G12, polC7,polD4K, PGV2, D4K enzyme, 9oN DNA polymerase, Tgo DNA polymerase, KodDNA polymerase or Deep vent DNA polymerase.

In one embodiment, the invention relates to a mXNAP display librarycomprising a population of display particles, wherein each displayparticle comprises a plurality of clonal nucleic acid moleculescomprising a dsDNA region and a single-stranded XNA aptamer region.

In one embodiment, the mXNAP library is a mTNAP, mHNAP, mCeNAP, mLNAP,mANAP, mphNAP, or mFANAP library.

In one embodiment, the mXNAP display library of comprises at least 10⁵mXNAPs.

In one embodiment, the invention relates to a method of screening for anXNA aptamer having a desired property, comprising the steps of: (a)incubating a mXNAP display library comprising a population of displayparticles, wherein each display particle comprises a plurality of clonalnucleic acid molecules comprising a dsDNA region and a single-strandedXNA aptamer region with at least one candidate interaction partner foran amount of time sufficient for interaction of the XNA aptamer regionswith the candidate interaction partner; and (b) selecting mXNAPparticles displaying XNA aptamers that have the desired property.

In one embodiment, the desired property is selected from the groupconsisting of a target binding activity and a target-binding inducedactivity.

In one embodiment, the invention relates to an XNA aptamer identifiedthrough screening a mXNAP library as having a desired property. In oneembodiment, the aptamer is a TNA aptamer, a HNA aptamer, a CeNA aptamer,a LNA aptamer, an ANA aptamer or a FANA aptamer.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising an XNA aptamer identified through screening a mXNAP libraryas having a desired property. In one embodiment, the composition furthercomprising a pharmaceutically acceptable excipient.

In one embodiment, the invention relates to a method of treating adisease or disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition comprising an XNA aptamer identified through screening amXNAP library as having a desired property.

In one embodiment, the invention relates to a TNA aptamer, comprising asequence selected from SEQ ID NO:1 to SEQ ID NO:3, a variant of SEQ IDNO:1 to SEQ ID NO:3, or a fragment comprising at least 20 nt of SEQ IDNO:1 to SEQ ID NO:3.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a TNA aptamer, comprising a sequence selected from SEQ IDNO:1 to SEQ ID NO:3, a variant of SEQ ID NO:1 to SEQ ID NO:3, or afragment comprising at least 20 nt of SEQ ID NO:1 to SEQ ID NO:3. In oneembodiment, the composition further comprising a pharmaceuticallyacceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 depicts schematic diagrams of the generation of monoclonal XNAaptamer particles in which many copies of the same XNA aptamer sequenceare displayed on the same bead, and the ensemble of beads constitutes alibrary of XNA aptamer particles for target screening.

FIG. 2 depicts a schematic diagram of the generation of XNA particledisplay molecules.

FIG. 3 depicts a schematic diagram of a screening method for detecting atarget protein using the XNA particle display molecules.

FIG. 4A through FIG. 4C depict the selection of High Affinity TNAAptamers. FIG. 4A depicts the chemical structure for the linearizedbackbone of RNA and α-L-threofuranosyl nucleic acid (TNA). FIG. 4Bdepicts an in vitro selection strategy designed to isolate TNA aptamersagainst HIV reverse transcriptase. A chemically synthesized DNA libraryencoding 40 random nucleotide positions flanked on both sides withfixed-sequence primer-binding sites is ligated onto a DNA stem-loop. Thepool of self-priming DNA templates is extended with tNTPs to produce apopulation of TNA-DNA hairpin structures. A separate DNA primer modifiedwith a 5′ IR dye is annealed to the loop region and extended with DNA todisplace the TNA strand. The resulting pool of TNA-dsDNA fusionmolecules is incubated with the target protein immobilized on Ni-NTAbeads, washed to remove non-functional molecules, recovered, andamplified by PCR. The dsDNA is made single-stranded and then used toinitiate another round of selection and amplification. FIG. 4C depictsMST binding affinity curves obtained for the top three TNA aptamersidentified after 3 rounds of in vitro selection. HIV-RT Aptamer 3.17 wasevaluated in triplicate. Error bars, standard deviation of each datapoint.

FIG. 5A through FIG. 5D, depicts the characterization of HIV-RT Aptamer3.17. FIG. 5A depicts the binding validation. The solution bindingaffinities (KD) of HIV-RT aptamer 3.17 and a known DNA aptamer werevalidated by native polyacrylamide gel electrophoresis. FIG. 5B depictsthe biological stability. Binding isotherms obtained in the presence ofSVPE for the TNA and DNA aptamers. FIG. 5C depicts the time-dependentnuclease stability profiles. IR-labeled DNA and TNA aptamers weremonitored by denaturing polyacrylamide gel electrophoresis followingexposure to SVPE. FIG. 5D depicts the thermal stability. HIV-RT aptamer3.17, a known DNA aptamer, and a commercial monoclonal antibody werechallenged to function at room temperature after heating at 70° C. for aperiod of 0-48 hours.

FIG. 6 depicts the structure activity relationship of HIV-RT aptamer3.17. MST derived KD values characterizing the binding of HIV-RT aptamer3.17 with deletions and mutations made to defined positions in themolecule.

DETAILED DESCRIPTION

In one embodiment, the present disclosure provides methods forgenerating an artificial nucleic acid, commonly referred to as XNAs or‘xeno-nucleic acids,’ particle display library of monoclonal XNA aptamerdisplay particles (mXNAPs), and the use of the display library to screenfor aptamers that bind to a target with high specificity.

In one embodiment, the present disclosure provides methods for use ofthe display library for identifying one or more unnatural nucleic acidagents, e.g., XNA aptamers, having a desired property identified from amixture of candidate XNA aptamers. The desired property may be a targetbinding activity or a target-binding induced activity, e.g., a catalyticactivity or a modified catalytic activity; inhibition activity,activation activity, or a modification of an inhibition activity oractivation activity; structure switching activity or a modification of astructure switching activity; or cooperative activity. In someembodiments, the desired property is a target binding activity or atarget-binding induced activity. In some embodiments, the target bindingactivity is one of affinity, specificity and bi-specificity.

XNA aptamers that can be displayed on the mXNAPs of the inventioninclude, but are not limited to, threose nucleic acid (TNA) aptamers,hexitol nucleic acid (HNA) aptamers, cyclohexene nucleic acid (CeNA)aptamers, locked nucleic acid (LNA) aptamers, arabino nucleic acid (ANA)aptamers, alkyl phosphonate nucleic acid (phNA) aptamers, and2′-deoxy-2′-fluoroarabinonucleic acid (FANA) aptamers.

In one embodiment, the invention relates to XNA aptamers identifiedusing the screening methods of the invention, compositions comprisingthe identified XNA aptamers and methods of use for the treatment of adisease or disorder.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassnon-limiting variations of ±40% or ±20% or ±10%, ±5%, ±1%, or ±0.1% fromthe specified value, as such variations are appropriate.

As used herein the term “aptamer” or “aptamer sequence” refers to anucleic acid having a specific binding affinity for a target, e.g., atarget molecule, wherein such target is other than a polynucleotide thatbinds to the aptamer or aptamer sequence through a mechanism whichpredominantly depends on Watson/Crick base pairing.

A “natural” nucleoside is one that occurs in nature. For the purposes ofthis invention the following nucleosides are defined as the naturalnucleosides: adenosine, cytidine, guanosine, uridine, 2′-deoxyadenosine,2′-deoxycytidine, 2′-deoxyguanosine, thymidine, and inosine.

The term base, unless otherwise specified, refers to the base moiety ofa nucleoside or nucleotide (a nucleobases). The base moiety is theheterocycle portion of a nucleoside or nucleotide. The base moiety maybe a pyrimidine derivative or analog, a purine derivative or analog, orother heterocycle. The nucleoside base may contain two or more nitrogenatoms and may contain one or more peripheral substituents. Thenucleoside base is attached to the sugar moiety of the nucleotide mimicin such ways that both β-D- and β-L-nucleoside and nucleotide can beproduced.

The term sugar refers to the ribofuranose of deoxyribofuranose portionof a nucleoside or nucleotide. The sugar moiety may contain one or moresubstituents at the C1-, C2-, C3-, C4-, and C5-position of theribofuranose. Substituents may direct to either the α- or β-face of theribofuranose. The nucleoside base that can be considered as asubstituent at the C-1 position of the ribofuranose directs to theβ-face of the sugar. The β-face is the side of a ribofuranose on which apurine or pyrimidine base of natural β-D-nucleosides is present. Theα-face is the side of the sugar opposite to the β-face. The sugar moietyof the present invention is not limited to a ribofuranose and itsderivatives, instead, it may be a carbohydrate, a carbohydrate analog, acarbocyclic ring, or other ribofuranose analog.

The term sugar-modified nucleoside refers to a nucleoside containing amodified sugar moiety.

The term base-modified nucleoside refers to a nucleoside containing amodified base moiety, relative to a base moiety found in a naturalnucleoside.

As used herein, the term “nucleic acid” refers to bothnaturally-occurring molecules such as DNA and RNA, but also variousderivatives and analogs. Generally, the probes, hairpin linkers, andtarget polynucleotides of the present teachings are nucleic acids, andtypically comprise DNA. Additional derivatives and analogs can beemployed as will be appreciated by one having ordinary skill in the art.

The term “nucleotide base”, as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, naturally occurring nucleotide bases adenine,guanine, cytosine, 6 methyl-cytosine, uracil, thymine, and analogs ofthe naturally occurring nucleotide bases, e.g., 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 delta2-isopentenyladenine (6iA), N6-delta 2-isopentenyl-2-methylthioadenine(2 ms6iA), N2-dimethylguanine (dmG), 7methylguanine (7mG), inosine,nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine,5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine,2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil,06-methylguanine, N6-methyladenine, 04-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see,e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT publishedapplication WO 01/38584), ethenoadenine, indoles such as nitroindole and4-methylindole, and pyrroles such as nitropyrrole. Certain exemplarynucleotide bases can be found, e.g., in Fasman, 1989, Practical Handbookof Biochemistry and Molecular Biology, pp. 385-394, CRC Press, BocaRaton, Fla., and the references cited therein.

The term “nucleotide”, as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR2 or halogen groups, where each Ris independently H, C1-C6 alkyl or C5-C14 aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-anomeric nucleotides, 1′-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352; andWO 99/14226). The term “nucleic acid” typically refers to largepolynucleotides.

The term “nucleotide analogs” as used herein refers to modified ornon-naturally occurring nucleotides including, but not limited to,analogs that have altered stacking interactions such as 7-deaza purines(i.e., 7-deaza-dATP and 7-deaza-dGTP); base analogs with alternativehydrogen bonding configurations (e.g., such as Iso-C and Iso-G and othernon-standard base pairs described in U.S. Pat. No. 6,001,983 to S.Benner and herein incorporated by reference); non-hydrogen bondinganalogs (e.g., non-polar, aromatic nucleoside analogs such as2,4-difluorotoluene, described by B. A. Schweitzer and E. T. Kool, J.Org. Chem., 1994, 59, 7238-7242; B. A. Schweitzer and E. T. Kool, J. Am.Chem. Soc., 1995, 117, 1863-1872); “universal” bases such as5-nitroindole and 3-nitropyrrole; and universal purines and pyrimidines(such as “K” and “P” nucleotides, respectively; P. Kong, et al., NucleicAcids Res., 1989, 17, 10373-10383, P. Kong et al., Nucleic Acids Res.,1992, 20, 5149-5152). Nucleotide analogs include nucleotides havingmodification on the sugar moiety, such as dideoxy nucleotides and2′-O-methyl nucleotides. Nucleoside analogue examples wherein thenatural sugar moiety is modified include but are not limited to hexitolnucleic acid (HNA), cyclohexene nucleic acids (CeNA), locked nucleicacids (LNA), altritol nucleic acids (ANA) and peptide nucleic acids(PNA). Nucleotide analogs include modified forms ofdeoxyribo-nucleotides as well as ribonucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning and amplification technology,and the like, and by synthetic means. An “oligonucleotide” as usedherein refers to a short polynucleotide, typically less than 100 basesin length.

As used herein, the term “pharmaceutical composition” refers to amixture of at least one compound useful within the invention with apharmaceutically acceptable carrier. The pharmaceutical compositionfacilitates administration of the compound to a patient or subject.Multiple techniques of administering a compound exist in the artincluding, but not limited to, intravenous, oral, aerosol, parenteral,ophthalmic, pulmonary and topical administration.

The term “reaction mixture” as used herein refers to a fluid medium inwhich a target is contacted with or in contact with candidate nucleicacid agents, e.g., candidate aptamer sequences. This includes, forexample, a reaction mixture in which a library of candidate nucleic acidagents, e.g., aptamer sequences, is initially contacted with a targetand any subsequent wash steps designed to remove non-specific orlow-affinity target binding agents. Where desired, the stringencyconditions of the reaction mixture can be modified so as to influencethe formation of complexes between the target and the candidate nucleicacid agents, e.g., candidate aptamer sequences. Thus, for example,stringency conditions of a reaction mixture during initial contacting oftarget and a library of candidate nucleic acid agents, e.g., candidateaptamer sequences, (which may be referred to as “binding conditions”)and stringency conditions of a reaction mixture during washing (referredto as “wash conditions”, e.g., to disrupt complexes of an undesirablylow affinity and/or deplete non-specifically bound candidate nucleicacid agents) may be of the same or different stringencies.

The term xeno-nucleic acid, abbreviated XNA, refers to a nucleic acidpolymer in which the natural ribose and deoxyribose sugars found in RNAand DNA replaced with another natural or unnatural sugar moiety.Examples include but are not limited to α-L-threofuranosyl nucleic acid(TNA), hexitol nucleic acid (HNA), arabino nucleic acid (ANA). XNAs arenot recognized by natural DNA and RNA polymerases, and therefore requireengineered polymerases for their synthesis.

The term aptamer particle, also known as monoclonal aptamer particle,refers to a bead that contains many copies of the same aptamer sequence.

The term XNA aptamer particle refers to an aptamer particle thatcontains many copies of the same XNA aptamer sequence.

The term XNA libraries refers to a combinatorial library of XNAmolecules having different sequence compositions.

The terms “specific binding,” “specifically bind,” and the like, referto the ability of a first binding molecule or moiety to preferentiallybind (covalently or non-covalently) to a second binding molecule ormoiety relative to other molecules or moieties in a reaction mixture.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, “TNA” or “TNAs” refer to nucleic acids having a backbonecomposed primarily of α-L-threofuranosyl-(3′→2′) (threose)-containingnucleotides, but may include heteropolymers comprising both tNTPs anddNTPs (e.g., dC).

As used herein, “tNTPs” refer to threose nucleotide triphosphates.

As used herein, “tNTP analog” refers to a threose nucleotidetriphosphate having a modified base moiety.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein, a sign or symptom of a condition contemplated herein or thepotential to develop a condition contemplated herein, with the purposeto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve oraffect a condition contemplated herein, the symptoms of a conditioncontemplated herein or the potential to develop a condition contemplatedherein. Such treatments may be specifically tailored or modified, basedon knowledge obtained from the field of pharmacogenomics.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation of asign, a symptom, or a cause of a disease or disorder, or any otherdesired alteration of a biological system. An appropriate therapeuticamount in any individual case may be determined by one of ordinary skillin the art using routine experimentation.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing an undesirable biological effect or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present disclosure provides methods for identifying one or moreunnatural nucleic acid agents, e.g., xeno-nucleic acid (XNA) aptamers,having a desired property identified from a mixture of candidate XNAaptamers. The method generally includes generating a population ofmonoclonal XNA display particles, wherein multiple clonal copies of asingle XNA aptamer are immobilized on any one of the particles, andwherein the population represents multiple unique monoclonal XNA displayparticles (mXNAPs). The particles are exposed to a target, and particlesincluding candidate nucleic acid agents having the desired property areisolated. In this way, one or more nucleic acid agents having thedesired property may be identified.

The desired property may be a target binding activity or atarget-binding induced activity, e.g., a catalytic activity or amodified catalytic activity; inhibition activity, activation activity,or a modification of an inhibition activity or activation activity;structure switching activity or a modification of a structure switchingactivity; or cooperative activity. In some embodiments, the desiredproperty is a target binding activity or a target-binding inducedactivity. In some embodiments, the target binding activity is one ofaffinity, specificity and bi-specificity.

The present disclosure also provides a quantitative, particle-basedmethod of generating and screening candidate XNA aptamers. Generally, alibrary of mXNAPs is prepared, wherein each mXNAPs displays multiplecopies of a unique candidate XNA aptamer sequence on its surface. ThemXNAPs are exposed to one or more target and each mXNAP is sorted basedon a quantitative analysis of an interaction between the candidateaptamer sequences on the mXNAP and the target. Following sorting, anenriched pool of mXNAPs may be provided which has reduced sequencediversity relative to the original library. One or more rounds ofscreening may be performed to identify XNA aptamers having desiredtarget interactions.

In one embodiment, the invention relates to methods of screening alibrary of XNA aptamers to identify aptamers having a desired property(e.g., high affinity for a target). XNA aptamers that can be displayedon the mXNAPs of the invention include, but are not limited to, threosenucleic acid (TNA) aptamers, hexitol nucleic acid (HNA) aptamers,cyclohexene nucleic acid (CeNA) aptamers, locked nucleic acid (LNA)aptamers, arabino nucleic acid (ANA) aptamers, glycerol nucleic acid(GNA), alkyl phosphonate nucleic acid (phNA) aptamers, and2′-deoxy-2′-fluoroarabinonucleic acid (FANA) aptamers. The inventionshould not be limited to the exemplary XNA's listed herein. Rather, theinvention includes any XNA including but not limited to XNA's currentlyavailable and XNA's that will become available.

In one embodiment, the invention relates to mXNAP display libraries foruse in the screening methods of the invention and to XNA aptamersidentified using the screening methods of the invention. In variousembodiments, the invention relates to pharmaceutical compositionscomprising the identified XNA aptamers and methods of use for thetreatment of a disease or disorder.

Nucleoside Triphosphates and Nucleic Acids

In one embodiment, the invention relates to nucleic acid moleculescontaining one or more nucleoside triphosphate analog or isomer (xNTPs).In one aspect, nucleoside triphosphate analogs or isomers can beincorporated into nucleic acid molecules including, but not limited tooligonucleotides, aptamers, deoxyribonucleic acid (DNA), ribonucleicacid (RNA) and peptide nucleic acid (PNA). A nucleic acid of theinvention also includes artificial nucleic acid polymers, commonlyreferred to as XNAs or ‘xeno-nucleic acids’ where the backbone structurecontains a sugar other than ribose or deoxyribose. While some of thesemolecules can be considered natural derivatives of RNA, like arabinonucleic acid (ANA), threose nucleic acid (TNA), and glycerol nucleicacid (GNA), others are completely unnatural, like locked nucleic acid(LNA), cyclohexene nucleic acid (CeNA), and hexitol nucleic acid (HNA).

Therefore, in one embodiment, the invention provides artificial orsynthetic nucleic acid molecules in which one or more nucleosidetriphosphate analog is incorporated. The length of the nucleic acids mayvary. The nucleic acids may be modified, e.g. may comprise one or moremodified nucleobases or modified sugar moieties (e.g., comprisingmethoxy groups). The backbone of the nucleic acid may comprise one ormore peptide bonds as in peptide nucleic acid (PNA). The nucleic acidmay comprise a base analog such as non-purine or non-pyrimidine analogor nucleotide analog. It may also comprise additional attachments suchas proteins, peptides and/or or amino acids.

In one embodiment, the XNA aptamers of the invention are TNA aptamers,containing one or more α-L-threofuranosyl nucleoside triphosphate(tNTP). tNTPs that can be included in a TNA aptamer of the inventioninclude, but are not limited to,1-(α-L-threofuranosyl)thymidine-3′-triphosphate (tTTP),1-(α-L-threofuranosyl)cytidine-3′-triphosphate (tCTP),9-(α-L-threofuranosyl)adenosine-3′-triphosphate (tATP), and9-(α-L-threofuranosyl)guanosine-3′-triphosphate (tGTP).

In various embodiments, the XNA aptamers of the invention are HNAaptamers, containing one or more 1′,5′-anhydrohexitol nucleosidetriphosphates (hNTPs), CeNA aptamers, containing one or morecyclohexenyl nucleoside triphosphate (CeNTP), LNA aptamers, containingone or more locked nucleic acid nucleoside triphosphates (1NTP), ANAaptamers, containing one or more arabino nucleoside triphosphates(1NTP), phNA aptamers, containing one or more P-α-ethylphosphonyl-β,γ-diphosphate nucleoside triphosphate (P-Et-phNTP) orP-α-methyl phosphonyl-β,γ-diphosphate (P-Met-phNTP), and FANA aptamers,containing one or more 2′-deoxy-2′-fluoroarabino nucleosidetriphosphates (faNTP). The invention should not be limited to theexemplary XNA's listed herein. Rather, the invention includes any XNAincluding but not limited to XNA's currently available and XNA's thatwill become available.

In one embodiment, the TNA aptamer of the invention comprises a TNAsequence as set forth in SEQ ID NO:1 through SEQ ID NO:3.

In some embodiments, the aptamer can be labeled. Examples of possiblelabels include, but are not limited to a radioisotope, an enzyme, anenzyme cofactor, an enzyme substrate, an enzyme inhibitor, a dye, ahapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a magnetic particle, an affinity label, a chromogenicagent, an azide group or other groups used for click chemistry, andother moieties known in the art.

TNA Oligonucleotides

In one embodiment, the invention provides biologically stable TNAoligonucleotides, wherein the TNA oligonucleotides comprise an effectiveamount of TNA and is completely resistant to enzymatic degradation. Asused herein, by “effective amount of TNA” means an amount of TNAsufficient to yield resistance to enzymatic degradation. In oneembodiment, the effective amount of TNA may comprise at least one TNAnucleic acid. In another embodiment, the effective amount of TNA maycomprise at least two TNA nucleic acids. In other embodiments, theeffective amount of TNA may comprise at least four TNA nucleic acids, atleast five TNA nucleic acids, at least six TNA nucleic acids, at leastseven TNA nucleic acids, at least ten nucleic acids.

As used herein, “resistant to enzymatic degradation” means the XNAoligonucleotide of the present invention resists degradation by enzymesincluding, without limitation, human liver microsomes, snake venomphosphodiesterase, RNAse A, RQ1 DNAse, and Turbo DNAse, for at least 24hours.

In one embodiment, the invention provides stable, nuclease-resistantTNA-DNA oligonucleotides, wherein the TNA-DNA oligonucleotides comprisean effective amount of TNA and is resistant to enzymatic degradation. Inone embodiment, the effective amount of TNA may comprise at least oneTNA nucleic acid.

In one embodiment, the TNA aptamer of the invention comprises a TNAsequence as set forth in SEQ ID NO:1 through SEQ ID NO:3 (Table 1), or afragment, derivative or variant thereof. In one embodiment, the TNAaptamer is a fragment comprising at least 20, 25, 30, 35 or at least 40nt of SEQ ID NO:1 through SEQ ID NO:3. In one embodiment, the TNAaptamer is a variant comprising a TNA sequence having at least 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to SEQ IDNO:1 through SEQ ID NO:3.

TABLE 1 HIV-RT binding TNA aptamers SEQ ID NO Sequence Description 1atagcaaattacttataaattagttcagta HIV-RT 3.10 gctactgtca 2agcaaagtccttggaatacgatcgtaccgt HIV-RT 3.15 tcctagacta 3aatagtaaattgatttaaaaatttcataaa HIV-RT 3.17 tgctacataa

In another embodiment, the invention provides a method of preparingnuclease-resistant TNA-DNA oligonucleotides, the method comprisingcontacting a self-priming, stem-loop forming template DNA molecule witha TNA polymerase and at least one tNTP in appropriate conditions suchthat the polymerase to synthesize nascent TNA from the priming end ofthe DNA molecule complementary to the template region of the DNAmolecule, to yield a TNA-DNA hybrid oligonucleotide.

In some embodiments, the effective amount of TNA in a TNA-DNAoligonucleotide is at least one TNA. In some embodiments, the effectiveamount of TNA in a TNA-DNA oligonucleotide is at least two TNA, at leastthree TNA, at least four TNA, at least 5 TNA, at least 6 TNA, at least 7TNA, at least 8 TNA, at least 9 TNA, at least 10 TNA, at least 15 TNA,at least 20 TNA, at least 25 TNA, at least 30 TNA, at least 35 TNA, atleast 40 TNA and may contain any number of TNA inbetween.

In some embodiments, the effective amount of TNA in a TNA-DNAoligonucleotide is at least 1% of the oligomers, at least 2% of theoligomers, at least 5%, or at least 7% of the oligomers. In someembodiments, the effective amount of TNA in the TNA-DNA oligonucleotideis at least 10% of the oligomers, at least 15% of the oligomers, atleast 20% of the oligomers, at least 25% of the oligomers, at least 30%of the oligomers, at least 35% of the oligomers, at least 40% of theoligomers, at least 50% of the oligomers, at least 60% of the oligomers,at least 70% of the oligomers, at least 80% of the oligomers and anyamounts or ranges inbetween (for example, 6%, 7%, 8%, 9%, 11%, 12%, 13%,14%, 15%, 17%, 18%, 19%, 21%, 22%, 23%, 24%, 26%, 26%, 27%, 28%, 29%,31%, 32%, 33%, 42%, 55%, 58%, 66% etc. etc.).

In another embodiment, the invention provides methods of using thenuclease-resistant TNA and TNA-DNA oligonucleotides of the presentinvention. The nuclease-resistant TNA and TNA-DNA oligonucleotides ofthe present invention may be used as a therapeutic (antisense, catalyst,RNAi etc), affinity reagent (aptamer, ribozyme) for diagnostic drugdelivery, diagnostic testing, imaging etc. In various embodiments, thenuclease-resistant TNA and TNA-DNA oligonucleotides of the presentinvention may be substituted in part or in whole for any applicationthat currently uses DNA or RNA.

mXNAP Display Library

Candidate nucleic acid agents and candidate aptamers for use in a screenof the invention may be provided in the form of an mXNAP display librarywhich includes a large number of display particles, wherein eachparticle is linked to a) template DNA molecules comprising a regionhaving a random nucleic acid sequence, which encodes an XNA aptamer, orb) dsDNA-XNA hybrid molecules wherein the XNA oligonucleotide comprisesan XNA aptamer. mXNAP display libraries may include, for example, fromabout 1×10² to about 1×10¹⁴ unique template DNA sequences or from about1×10² to about 1×10¹⁴ unique candidate XNA aptamer sequences, e.g., fromabout 1×10³ to about 1×10¹⁴ unique sequences, from about 1×10⁴ to about1×10¹⁴ unique sequences, from about 1×10⁵ to about 1×10¹⁴ uniquesequences, from about 1×10⁶ to about 1×10¹⁴ unique sequences, from about1×10⁷ to about 1×10¹⁴ unique sequences, from about 1×10⁸ to about 1×10¹⁴unique sequences, from about 1×10⁹ to about 1×10¹⁴ unique sequences,from about 1×10¹⁰ to about 1×10¹⁴ unique sequences, from about 1×10¹¹ toabout 1×10¹⁴ unique sequences, from about 1×10¹² to about 1×10¹⁴ uniquesequences, or from about 1×10¹³ to about 1×10¹⁴ unique sequences.

XNA aptamer encoding sequences or XNA aptamers displayed on the mXNAPsof the invention may be, for example, from about 30 to about 150nucleotides in length, e.g., from about 40 to about 130 nucleotides inlength, from about 50 to about 120 nucleotides in length, from about 60to about 110 nucleotides in length, from about 70 to 100 nucleotides inlength, or from about 80 to about 90 nucleotides in length. Candidatenucleic acid agents and candidate aptamers including nucleic acidsequences may include random nucleic acid sequences of from about 30nucleotides in length to about 70 nucleotides in length, e.g., fromabout 40 nucleotides in length to about 60 nucleotides in length. Inaddition to random nucleic acid sequence regions, template DNA sequencesand XNA sequences may may include flanking regions containing primerbinding sites.

Particles

A variety of suitable particles may be used in the generation of themXNAP display library as described herein. Such particles may be sizedto have at least one dimension, e.g., diameter, of from about 50 nm toabout 100 For example, in some embodiments a suitable particle is sizedto have at least one dimension of from about 50 nm to about 1 μm, e.g.,from about 50 nm to about 500 nm, or from about 50 nm to about 100 nm.In other embodiments, a suitable particle is sized to have at least onedimension of from about 500 nm to about 100 μm, e.g., from about 1 μm toabout 100 or from about 50 μm to about 100 Suitable particles may begenerally spherical or may have any other suitable shape.

Particles may be made from a variety of suitable materials known in theart. For example, magnetic particles may be utilized in the disclosedmethods and compositions. Suitable magnetic particles may include, forexample, magnetic beads or other small objects made from a magneticmaterial such as a ferromagnetic material, a paramagnetic material, or asuperparamagnetic material. Magnetic particles may include, e.g., ironoxide (Fe₂O₃ and/or Fe₃O₄). Additional particles of interest may includepolymer-based particles, e.g., polymer based beads. For example,polystyrene particles may be utilized. In addition, in some embodimentsceramic particles may be utilized.

The particles may include or be coated with a material which facilitatescoupling of the particles to candidate nucleic acid agents and candidateaptamer sequences. Examples of coatings include polymer shells, glasses,ceramics, gels, etc. In some embodiments, the coatings include or arethemselves coated with a material that facilitates coupling or physicalassociation of the particles with the candidate nucleic acid agentsequences and candidate aptamer sequences. For example, particles withexposed carboxylic acid groups may be used for attachment to candidatenucleic acid agents and candidate aptamers.

Suitable particles may include one or more functional groups, e.g., oneor members of a reactive pair as described herein, positioned on one ormore surfaces of the particles. Suitable functional groups may include,for example, amine groups, carboxyl groups, thiol groups, SiO₂, EDTA,and boronic acid functional groups.

In some embodiments, suitable particles may include one or more membersof a specific binding pair on one or more surfaces of the particles. Forexample, avidin, streptavidin, Neutravidin®, Captavidin™, or biotin maybe positioned on one or more surfaces of the particles.

Methods of Attaching Candidate Nucleic Acid Agents and Candidate AptamerSequences to Particles

A variety of methods may be used to attach nucleic acid molecules (e.g.,primer sequences), to particles for use in generating mXNAPs asdescribed herein.

In one suitable method nucleic acid molecules may be attached to aparticle having exposed carboxylic acid groups using amino groupmodification. For example, 5′-amino modified oligonucleotides may beused in connection with carbodiimide mediated amide bond formation toattach the oligonucleotide sequences to particles.

Carbodiimide mediated coupling methods are described in greater detail,for example, in Nakajima N. and Ikade Y. (1995) Bioconjugate Chem.,6(1):123-130; Gilles et al. (1990) Anal Biochem., 184(2):244-248; SehgalD. and Vijay 1K. (1994) Anal Biochem. 218(1):87-91; and Szajani et al.(1991) Appl Biochem Biotechnol. 30(2):225-231.

DNA Display

DNA display is a method in which XNA-DNA fusion molecules are generatedin which a newly transcribed XNA oligonucleotide is linked to itscorresponding DNA template. This is achieved by using a self-primingstem-loop forming DNA molecule which is ligated to a single stranded DNAmolecule comprising a template region for the synthesis of the desiredXNA oligonucleotide.

When the DNA display template is replicated, xNTPs are incorporated intothe newly-synthesized portion of the molecule by an XNA polymerase. TheXNA region is then displaced from the template molecule resulting in aDNA-XNA fusion molecule.

In one embodiment, tNTPs are incorporated into the newly-synthesizedportion of the molecule by a TNA polymerase. The TNA region is thendisplaced from the template molecule resulting in a DNA-TNA fusionmolecule. Exemplary TNA polymerases that can be used in the methods ofthe invention include, but are not limited to, Kod-RSGA, Kod-RS, Kod-RI,and Therminator TNA polymerase.

In one embodiment, hNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing HNA froma DNA template. The HNA region is then displaced from the templatemolecule resulting in a DNA-HNA fusion molecule. Exemplary polymerasescapable of synthesizing HNA from a DNA template that can be used in themethods of the invention include, but are not limited to, pol6G12_521Lpolymerase.

In one embodiment, CeNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing CeNAfrom a DNA template. The CeNA region is then displaced from the templatemolecule resulting in a DNA-CeNA fusion molecule. Exemplary polymerasescapable of synthesizing CeNA from a DNA template that can be used in themethods of the invention include, but are not limited to, pol6G12, polC7and polD4K polymerase.

In one embodiment, 1NTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing LNA froma DNA template. The LNA region is then displaced from the templatemolecule resulting in a DNA-LNA fusion molecule. Exemplary polymerasescapable of synthesizing LNA from a DNA template that can be used in themethods of the invention include, but are not limited to, polC7 andpolD4K polymerase.

In one embodiment, aNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing ANA froma DNA template. The ANA region is then displaced from the templatemolecule resulting in a DNA-ANA fusion molecule. Exemplary polymerasescapable of synthesizing ANA from a DNA template that can be used in themethods of the invention include, but are not limited to, polC7 andpolD4K polymerase.

In one embodiment, phNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing ANA froma DNA template. The phNA region is then displaced from the templatemolecule resulting in a DNA-phNA fusion molecule. Exemplary polymerasescapable of synthesizing phNA from a DNA template that can be used in themethods of the invention include, but are not limited to, PGV2polymerase.

In one embodiment, faNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing FANAregion is then displaced from the template molecule resulting in aDNA-FANA fusion molecule. Exemplary polymerases capable of synthesizingFANA from a DNA template that can be used in the methods of theinvention include, but are not limited to, D4K enzyme, 9oN DNApolymerase, Tgo DNA polymerase, Kod DNA polymerase and Deep vent DNApolymerase.

The DNA from a selected DNA-XNA fusion can be amplified, and ligated toa hairpin forming DNA molecule ready for the next round of selection.The ability to carry out multiple rounds of selection and amplificationenables the enrichment and isolation of very rare molecules, e.g., onedesired molecule out of a pool of 10¹³ members. This in turn allows theisolation of new or improved XNA or XNA aptamers which can specificallyrecognize a target or which can catalyze desired chemical reactions.

In one aspect, described herein is a method of making a DNA displaylibrary displaying TNA, HNA, CeNA, LNA, ANA, phNA or FANA aptamers, or acombination thereof. In certain embodiments, the DNA display librarycomprises molecules for the display of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³, or at least 10¹⁴ different XNA aptamers.

In one aspect, provided herein is a method of preparing a DNA displaylibrary comprising the steps of: (a) providing a population ofself-priming, stem-loop forming, template DNA molecules, each of whichcomprises a stem-loop forming priming sequence, a random DNA codingregion and a fixed sequence primer binding site; (b) extending theprimer in the presence of an XNA polymerase and one or more XNAtriphosphate molecules (xNTPs) to form a population of double strandedXNA-DNA display templates; and (c) contacting the double strandedXNA-DNA display templates with a primer which anneals to the loop regionof the stem-loop structure and extending the DNA primer using dNTPs anda DNA polymerase to displace the XNA portion of the XNA-DNA displaytemplates to form a population of display molecules comprising a dsDNAregion and a single-stranded XNA region (dsDNA-ssXNA fusion displaymolecule).

Particle Display

In one embodiment, the invention provides a method of generating amonoclonal XNA-particle (mXNAP) display library. In one embodiment, themXNAPs of the invention are generated by coupling the 5′ end of a singlestranded DNA molecule comprising a DNA template region for the synthesisof the desired XNA oligonucleotide.

In one embodiment, the template DNA molecule can be attached to theparticles using emulsion PCR methods prior to ligation to the hairpinforming DNA molecule. Generally, emulsion PCR as used in connection withthe disclosed methods isolates individual template DNA molecules, e.g.,from a combinatorial library, along with primer-coated particles, e.g.,beads, in aqueous droplets within an oil phase. PCR amplification thencoats each bead with clonal copies of the DNA molecule. After breakingthe emulsion and removing unreacted PCR reagents, hybridized strands maybe de-hybridized and the template particles collected for subsequentligation to a self-priming DNA hairpin molecule. In one embodiment, theXNA is then generated by contacting the ligated template particles witha reaction mixture containing at least one xNTP and at least onepolymerase capable of synthesizing XNA from a DNA template (an XNApolymerase).

When the DNA display template is copied, xNTPs are incorporated into thenewly-synthesized portion of the molecule by the XNA polymerase. The XNAregion is then displaced from the template molecule resulting in aDNA-XNA fusion molecule coupled to the particle.

In one embodiment, tNTPs are incorporated into the newly-synthesizedportion of the molecule by a TNA polymerase. The TNA region is thendisplaced from the template molecule resulting in a DNA-TNA fusionmolecule. The resultant display library is a mTNAP library. ExemplaryTNA polymerases that can be used in the methods of the inventioninclude, but are not limited to, Kod-RSGA, Kod-RS, Kod-RI, andTherminator TNA polymerase.

In one embodiment, hNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing HNA froma DNA template. The HNA region is then displaced from the templatemolecule resulting in a DNA-HNA fusion molecule. The resultant displaylibrary is a mHNAP library. Exemplary polymerases capable ofsynthesizing HNA from a DNA template that can be used in the methods ofthe invention include, but are not limited to, pol6G12_521L polymerase.

In one embodiment, CeNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing CeNAfrom a DNA template. The CeNA region is then displaced from the templatemolecule resulting in a DNA-CeNA fusion molecule. The resultant displaylibrary is a mCeNAP library. Exemplary polymerases capable ofsynthesizing CeNA from a DNA template that can be used in the methods ofthe invention include, but are not limited to, pol6G12, polC7 and polD4Kpolymerase.

In one embodiment, 1NTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing LNA froma DNA template. The LNA region is then displaced from the templatemolecule resulting in a DNA-LNA fusion molecule. The resultant displaylibrary is a mLNAP library. Exemplary polymerases capable ofsynthesizing LNA from a DNA template that can be used in the methods ofthe invention include, but are not limited to, polC7 and polD4Kpolymerase.

In one embodiment, aNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing ANA froma DNA template. The ANA region is then displaced from the templatemolecule resulting in a DNA-ANA fusion molecule. The resultant displaylibrary is a mANAP library. Exemplary polymerases capable ofsynthesizing ANA from a DNA template that can be used in the methods ofthe invention include, but are not limited to, polC7 and polD4Kpolymerase.

In one embodiment, phNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing phNAfrom a DNA template. The phNA region is then displaced from the templatemolecule resulting in a DNA-phNA fusion molecule. The resultant displaylibrary is a mphNAP library. Exemplary polymerases capable ofsynthesizing phNA from a DNA template that can be used in the methods ofthe invention include, but are not limited to, PGV2 polymerase.

In one embodiment, faNTPs are incorporated into the newly-synthesizedportion of the molecule by a polymerase capable of synthesizing FANAregion is then displaced from the template molecule resulting in aDNA-FANA fusion molecule. The resultant display library is a mFANAPlibrary. Exemplary polymerases capable of synthesizing FANA from a DNAtemplate that can be used in the methods of the invention include, butare not limited to, D4K enzyme, 9oN DNA polymerase, Tgo DNA polymerase,Kod DNA polymerase and Deep vent DNA polymerase.

In one aspect, described herein are mXNAP display libraries displayingTNA, HNA, CeNA, LNA, ANA, pHNA or FANA aptamers, or combinationsthereof. In certain embodiments, the mXNAP display library comprisesmolecules for the display of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, or at least 10¹⁴ different XNA aptamers.

In one aspect, provided herein is a method of preparing a XNA particledisplay library comprising the steps of: (a) providing a population ofmonoclonal particles, wherein each particle comprises a plurality ofself-priming, stem-loop forming, template DNA molecules, each of whichcomprises a stem-loop forming priming sequence, a DNA coding region anda fixed sequence primer binding site; (b) extending the primer in thepresence of an XNA polymerase and one or more XNA triphosphate molecules(xNTPs) to form a population of double stranded XNA-DNA hybridparticles; and (c) contacting the double stranded XNA-DNA hybridparticles with a primer which anneals to the loop region of thestem-loop structure and extending the DNA primer using dNTPs and a DNApolymerase to displace the XNA portion of the XNA-DNA display templatesto form a population of display particles comprising pluralitydsDNA-ssXNA fusion display molecules.

Screening Methods

In one aspect, provided herein is a method of screening for XNA aptamershaving a desired property comprising: (i) incubating a mXNAP displaylibrary containing XNA aptamers with at least one candidate interactionpartner for an amount of time sufficient for interaction (e.g., binding)of the XNA aptamers with the candidate interaction partner; (ii) washingto remove the unbound XNA aptamers; (iii) selecting XNA aptamers thathave the desired property.

The desired property may be a target binding activity or atarget-binding induced activity, e.g., a catalytic activity or amodified catalytic activity; inhibition activity, activation activity,or a modification of an inhibition activity or activation activity;structure switching activity or a modification of a structure switchingactivity; or cooperative activity. In some embodiments, the desiredproperty is a target binding activity or a target-binding inducedactivity. In some embodiments, the target binding activity is one ofaffinity, specificity and bi-specificity.

In one such embodiment, the target binding activity is specificity, andthe screening method includes a step of exposing the plurality of mXNAPto a first target and a second target. Nucleic acid agents having thedesired property will exhibit a specific binding affinity (e.g, a Kd offrom about 1 pM to about 100 nM, e.g., a Kd of from about 1 pM to about10 nM, or a Kd of from about 1 pM to about 5 nM) for either the firsttarget or the second target but not both. For example, the first targetmay be a first homolog or splicing variant of a protein and the secondtarget may be a second homolog or splicing variant of the protein. Inanother embodiment, the first target may be a first post-translationalmodification form of a protein and the second target may be a secondpost-translation modification form of a protein. In another embodiment,the first target may be a protein which has been subjected to apost-translational modification and the second target may be a form ofthe protein which has not been subjected to the post-translationalmodification. For example, a nucleic acid agent having the desiredproperty may bind to a phosphorylated form of a protein but not theunphosphorylated form or vice versa.

A variety of post-translational modifications are known in the art,e.g., myristoylation, palmitoylation, isoprenylation or prenylation,glypiation, lipoylation, the addition of flavin, the addition of heme C,phosphopantetheinylation, retinylidene Schiff base formation, acylation(e.g., acetylation), alkylation (e.g., methylation), amide bondformation, glycosylation, nucleotide addition, oxidation, phosphateester (O-linked) or phosphoramidate (N-linked) formation (e.g.,phosphorylation and adenylylation), glycation, biotinylation andPEGylation, among others.

In some embodiments, the first target is a first conformational form ofa protein and the second target is a second conformational form of theprotein. For example, the first target may be a ligand-bound form of anenzyme and the second target may be an unbound form of the same enzymeor vice versa.

In some embodiments, the target binding activity is bi-specificity. Insuch embodiments, the screening method may include a step of exposingthe plurality of display molecules to a first target and a secondtarget. Nucleic acid agents having the desired property will exhibit aspecific binding affinity (e.g, a Kd of from about 1 pM to about 100 nM,e.g., a Kd of from about 1 pM to about 10 nM, or a Kd of from about 1 pMto about 5 nM) for both the first and second target. In suchembodiments, the first target may be a first homolog or splicing variantof a protein and the second target may be a second homolog or splicingvariant of the protein. The first target may be a firstpost-translational modification form of a protein and the second targetmay be a second post-translation modification form of a protein, e.g.,as described above. In addition, the first target may be a protein whichhas been subjected to a post-translational modification and the secondtarget may be a form of the protein which has not been subjected to thepost-translational modification, e.g., as described above. The firsttarget may be a first conformational form of a protein and the secondtarget may be a second conformational form of the protein, e.g., asdescribed above.

In some embodiments, multiple detectable labels may be used tofacilitate the screening process. For example, where the target bindingactivity is specificity, the screening method may include exposing theplurality of display molecules to a first target labeled with a firstdetectable label and a second target labeled with a second detectablelabel, wherein the first detectable label and the second detectablelabel are different. Nucleic acid agents having the desired propertywill exhibit a first binding affinity for the first target and a secondbinding affinity for the second target, wherein the first bindingaffinity and the second binding affinity are significantly different.These binding affinities may be determined via detection of thedetectable labels. For example, in order to screen for aptamers thatspecifically bind to a thrombin protein in serum, thrombin can belabeled with a first detectable label while all other serum proteins arelabeled with a second, different detectable label. Aptamers associatedwith a relatively high signal from the first detectable label, which isindicative of relatively high affinity thrombin binding, and arelatively low signal from the second detectable label, which isindicative of relatively low affinity binding to other serum proteins,may be selected.

Similarly, multiple detectable labels may be used where the targetbinding activity is bi-specificity. For example, the screening methodmay include exposing the plurality of display molecules to a firsttarget labeled with a first detectable label and a second target labeledwith a second detectable label, wherein the first detectable label andthe second detectable label are different. Nucleic acid agents havingthe desired property will exhibit a specific binding affinity (e.g, a Kdof from about 1 pM to about 100 nM, e.g., a Kd of from about 1 pM toabout 10 nM, or a Kd of from about 1 pM to about 5 nM) for both thefirst and second target, which binding affinity may be determined viadetection of the detectable labels.

Isolation and/or sorting as described herein may be conducted using avariety of methods and/or devices known in the art, e.g., flow cytometry(e.g., Fluorescence Activated Cell Sorting (FACS) or Ramen flowcytometry), fluorescence microscopy, optical tweezers, micro-pipettes,and microfluidic magnetic separation devices and methods. In someembodiments, where the detectably labeled target is a fluorescentlylabeled target, Fluorescence Activated Cell Sorting (FACS) may beutilized to quantitatively sort particle immobilized candidate nucleicacid agents or aptamer particles based on one or more fluorescencesignals. One or more sort gates or threshold levels may be utilized inconnection with one or more detectable labels to provide quantitativesorting over a wide range of candidate nucleic acid agent-targetinteractions or candidate aptamer sequence-target interactions. Inaddition, the screening stringency may be quantitatively controlled,e.g., by modulating the target concentration and setting the position ofthe sort gates.

Where, for example, the fluorescence signal is related to the bindingaffinity of the candidate nucleic acid agents or candidate aptamersequences for the target, the sort gates and/or stringency conditionsmay be adjusted to select for nucleic acid agents or aptamers having adesired affinity or desired affinity range for the target. In somecases, it may be desirable to isolate the highest affinity nucleic acidagents or aptamers from a particular library of candidate nucleic acidagents or candidate aptamer sequences. However, in other cases nucleicacid agents or aptamers falling within a particular range of bindingaffinities may be isolated.

Targets

Candidate nucleic acid agents and aptamers may be generated and screenedas described herein to identify nucleic acid agents and aptamers whichbind to a variety of targets, e.g., target molecules. Suitable targetsmay include, for example, small molecules (e.g., organic dyes, toxins,etc.), amino acids, carbohydrates, lipids, aminoglycosides, antibiotics,peptides, proteins, post-translational modifications, nucleic acids,liposomes, virus, whole cells, cellular components, tissues, livingorganisms, or an unknown target or mixture. Small molecule targets ofinterest generally have a molecular weight of about 1000 Daltons, e.g.,less than 800 Daltons. Protein targets of interest may include, forexample, cell surface receptors, signal transduction factors, andhormones. Cellular targets of interest may include, for example,mammalian cells, particularly human cells; stem cells; tumor cells andbacterial cells.

More than one type of target may be utilized simultaneously in thescreening methods disclosed herein. For example, two or more proteintargets having different amino acid sequences may be simultaneouslyscreened against a single library of candidate nucleic acid agents orcandidate aptamer sequences.

In some embodiments, a target molecule or a molecule associated with atarget molecule, e.g., via a binding interaction, may be detectablylabeled as described herein.

Labels

Suitable labels which may be used to provide a detectably labeled targetor detectably labeled nucleic acid agent, e.g., aptamer, according tothe present disclosure may include radioactive isotopes, fluorescers,chemiluminescers, chromophores, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands(e.g., biotin, avidin, strepavidin or haptens), affinity tags and thelike.

Exemplary affinity tags suitable for use include, but are not limitedto, a monocytic adaptor protein (MONA) binding peptide, a T7 bindingpeptide, a streptavidin binding peptide, a polyhistidine tract, proteinA (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., MethodsEnzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson,Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al., Proc.Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hopp etal., Biotechnology 6:1204 (1988)), or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

Any fluorescent polypeptide (also referred to herein as a fluorescentlabel) may be suitable for use as a detectable label. A suitablefluorescent polypeptide will be one that will readily provide adetectable signal that can be assessed qualitatively (positive/negative)and quantitatively (comparative degree of fluorescence). Exemplaryfluorescent polypeptides include, but are not limited to, yellowfluorescent protein (YFP), cyan fluorescent protein (CFP), GFP, mRFP,RFP (tdimer2), HCRED, etc., or any mutant (e.g., fluorescent proteinsmodified to provide for enhanced fluorescence or a shifted emissionspectrum), analog, or derivative thereof. Further suitable fluorescentpolypeptides, as well as specific examples of those listed herein, areprovided in the art and are well known.

Biotin-based labels also find use in the methods disclosed herein.Biotinylation of target molecules is well known, for example, a largenumber of biotinylation agents are known, including amine-reactive andthiol-reactive agents, for the biotinylation of proteins, nucleic acids,carbohydrates, carboxylic acids; see, e.g., chapter 4, Molecular ProbesCatalog, Haugland, 6th Ed. 1996, hereby incorporated by reference. Abiotinylated substrate can be detected by binding of a detectablylabeled biotin binding partner, such as avidin or streptavidin.Similarly, a large number of haptenylation reagents are also known.

Pharmaceutical Compositions

In some embodiments, XNA aptamers identified according to the methods ofthe invention can function as pharmaceutical agents. For example, in oneembodiment, HIV-RT 3.17 can be used as an anti-HIV agent for thetreatment or prevention of HIV or an HIV associated disease or disorder.

Therefore, in one embodiment, the invention also relates to apharmaceutical composition comprising a therapeutically effective amountof an XNA aptamer of the invention, optionally in combination with oneor more other active ingredients and/or with a pharmaceuticallyacceptable carrier. Moreover, XNA aptamers of the invention may be usedin a method for the treatment of a disease or disorder, comprisingadministering a therapeutically effective amount of the XNA aptamer ofthe invention to a subject in need thereof.

The pharmaceutical composition of the present invention comprises atleast one XNA aptamer selected according to the methods of theinvention. The compositions include those suitable for oral, topical,intravenous, subcutaneous, nasal, ocular, pulmonary, and rectaladministration. The compounds of the invention can be administered tomammalian individuals, including humans, as therapeutic agents.

For example, the compounds of the invention are useful as antiviralagents. The present invention provides a method for the treatment of apatient afflicted with a viral infection comprising administering to thepatient a therapeutically effective antiviral amount of a compound ofthe invention. The term “viral infection” as used herein refers to anabnormal state or condition characterized by viral transformation ofcells, viral replication and proliferation. In one embodiment, the viralinfection is an HIV infection and the XNA aptamer of the invention isHIV-RT 3.17.

A “therapeutically effective amount” of a compound of the inventionrefers to an amount which is effective, upon single or multiple doseadministration to the patient, in controlling the growth of e.g., themicrobe or tumor or in prolonging the survivability of the patientbeyond that expected in the absence of such treatment. As used herein,“controlling the growth” refers to slowing, interrupting, arresting orstopping the microbial or proliferative transformation of cells or thereplication and proliferation of the microbe and does not necessarilyindicate a total elimination of e.g., the microbe or tumor.

Accordingly, the present invention includes pharmaceutical compositionscomprising, as an active ingredient, at least one of the compounds ofthe invention in association with a pharmaceutical carrier. Thecompounds of this invention can be administered by oral, parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), topical, transdermal (either passively or usingiontophoresis or electroporation), transmucosal (e.g., nasal, vaginal,rectal, or sublingual) or pulmonary (e.g., via dry powder inhalation)routes of administration or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating, agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, with the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

Topical formulations will generally comprise ointments, creams, lotions,gels or solutions. Ointments will contain a conventional ointment baseselected from the four recognized classes: oleaginous bases;emulsifiable bases; emulsion bases; and water-soluble bases. Lotions arepreparations to be applied to the skin or mucosal surface withoutfriction, and are typically liquid or semiliquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and preferably,for the present purpose, comprise a liquid oily emulsion of theoil-in-water type. Creams, as known in the art, are viscous liquid orsemisolid emulsions, either oil-in-water or water-in-oil. Topicalformulations may also be in the form of a gel, i.e., a semisolid,suspension-type system, or in the form of a solution.

Formulations of these drugs in dry powder form for delivery by a drypowder inhaler offers yet another means of administration. Thisovercomes many of the disadvantages of the oral and intravenous routes.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally, dosage levels of between 0.001 to 10 mg/kg of body weightdaily are administered to mammals.

Kits

The present invention also relates to a kit for performing any of theabove described methods. In one embodiment, the kit comprises at leastone reagent for use in a method of generating a mXNAP display library ofthe invention. In one embodiment, the kit may comprise a mixture ofxNTPs and an XNA polymerase for the synthesis of a mXNAP display libraryof the invention. In one embodiment, the kit may comprise an mXNAPdisplay library of the invention. In some embodiments, one or more ofthe components are premixed in the same reaction container. Inparticular embodiments, the kit additionally comprises instructionalmaterial.

In one embodiment, the kit comprises at least one XNA aptamer identifiedaccording to a method of the invention and instructions for use of theXNA aptamer. In one embodiment, the XNA aptamer is a TNA aptamer. In oneembodiment, the XNA aptamer is HIV-RT 3.17.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless so specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein. Without further description, itis believed that one of ordinary skill in the art can, using thepreceding description and the following illustrative examples, make andutilize the compounds of the present invention and practice the claimedmethods. The following working examples therefore, specifically pointout the preferred embodiments of the present invention, and are not tobe construed as limiting in any way the remainder of the disclosure

Example 1: mXNAP Display

A method of displaying XNA aptamers as a particle display library hasbeen developed. This method uses a modified version of DNA display,which is a strategy for covalently linking newly synthesized XNA strandsto their encoding double-stranded DNA, to allow construction of XNAaptamer particles. Schematic diagrams detailing the strategy areprovided in FIG. 1 through FIG. 3. For the generation of an XNA particledisplay library, a fixed primer sequence is coupled to a bead.Monoclonal beads are then generated to have a single unique template DNAsequence encoding an XNA aptamer using emulsion PCR. The emulsion isthen broken and the and complementary strands are removed prior toligation of a self-priming stem-loop forming DNA molecule to theparticle-bound template DNA sequence. Synthesis by a XNA polymerase fromthe self-priming end of the stem-loop forming DNA molecule thenincorporates xNTPs to form a DNA-XNA hybrid molecule. Stranddisplacement of the XNA aptamer using a primer that binds to thestem-loop forming molecule then generates monoclonal particle bounddsDNA-XNA display molecules.

Example 2: Generating Biologically Stable TNA Aptamers that Functionwith High Affinity and Thermal Stability

Although antibodies remain the gold standard for protein affinityreagents, they also represent the greatest source of problems inbiomedical research (Baker et al., 2015, Nature 521:274-276). Thesensitivity of antibodies to elevated temperatures demonstrates acritical weakness that limits their shelf-life, reproducibility, andperformance in functional assays. Thermal instability could be linked toproblems with batch-to-batch variability that have plagued antibodyresearch. In one striking example, the results from only six of 53 highprofile cancer research papers could be reproduced (Begley and Ellis,2012, 2012, Nature 483:531-533). In another study, 25% of 246 antibodiesused for epigenetic research failed tests for specificity (Egelhofer etal., 2011, Nat. Struct. Mol. Biol. 18, 91-93). Similarly, 49 antibodiesgenerated against 19 signaling proteins had bound to more than onetarget, meaning that their results could not be trusted 34. Given theseproblems, researchers are calling for better standardization methods andaccess to new types of affinity reagents (Bradbury and Pluckthun, 2015,Nature, 518:27-29; Marx et al., 2013, Nat. Methods 10:29-833).

The experiments presented herein outline a selection approach forevolving biologically stable aptamers based on the framework ofα-L-threofuranosyl nucleic acid (TNA, FIG. 4A) (Schöning et al., 2000,Science, 290:1347-1351) an artificial genetic system discovered byEschenmoser and colleagues as part of a systematic investigation intothe chemical etiology of RNA (Eschenmoser, 1999, Science,284:2118-2124). Despite a backbone repeat unit that is one atom (orbond) shorter than its natural counterpart, TNA is capable of efficientWatson-Crick base pairing with itself and with complementary strands ofDNA and RNA (Schöning et al., 2000, Science, 290:1347-1351; Yang et al.,2007, J. Mol. Evol. 65:289-295). More recently, biological studies haveshown that TNA is completely refractory to nuclease digestion, making ita promising candidate for biological applications that requiretarget-specific binding in environments where natural genetic polymersrapidly degrade (Culbertson et al., 2016, Bioorg. Med. Chem. Lett.26:2418-2421). The strategy taken is analogous to protein displaytechnologies, such as mRNA display, that provide a covalent link betweenthe encoding messenger RNA template (genotype) and translated protein(phenotype) (Roberts and Szostak, 1997, Proc. Natl. Acad. Sci. USA94:12297-12302). However, in this case, freshly synthesized TNA isphysically linked to its complementary DNA template, which is present indouble-stranded (ds) form (FIG. 4B) (Ichida et al., 2005, J. Am. Chem.Soc. 127:2802-2803). In this configuration, TNA molecules isolated fromthe selection are amplified by PCR using the dsDNA portion of themolecule as the template for the polymerase. This approach issufficiently general that it could be applied to any XNA polymer(artificial genetic polymers with non-ribose sugars) for which apolymerase is available to copy DNA templates into XNA (Chaput et al.,2012, Chem. Biol. 19, 1360-1371). It also avoids the need for an XNAreverse transcriptase, which are difficult to generate by polymeraseengineering and tend to function with weak template binding affinity.The latter problem is particularly acute, as it can limit the recoveryof functional sequences when small numbers of XNA molecules are presentafter stringent washing steps have been carried out to remove weakeraffinity sequences.

Although substantially more work is needed, including access to buildingblocks with greater chemical diversity, the data suggests thatalternative protein affinity reagents, like TNA aptamers, may offercertain advantages to antibodies. Unlike most aptamers, TNA iscompletely recalcitrant to nuclease digestion and is amenable to invitro selection against any biological protein of therapeutic ordiagnostic interest. The latter provides an important benefit overSpiegelmers, whose targets must be generated by chemical synthesis.Relative to antibodies, TNA is capable of achieving KDs with picomolarbinding while also avoiding unwanted problems associated with thermaldenaturation. The ability to fold cooperatively into functionalstructures, combined with their chemical synthesis, solves thecold-chain problem and could improve the reproducibility and performanceof protein affinity reagents.

The results presented herein establish that TNA aptamers have theability to function with high biological stability, protein bindingaffinity, and thermal stability. These data offer a possible solution tothe antibody problem and provide strong support for the continueddevelopment of TNA reagents for diagnostic and therapeutic applications.Such projects open the door to a new generation of affinity reagentsthat could one day overcome some of the weaknesses of existingtechnologies.

The results of the experiments are now described.

The selection (FIG. 4B) was performed by extending a self-priming DNAlibrary with chemically synthesized TNA triphosphates (tNTPs) (Sau etal., 2016, J. Org. Chem. 81:2302-2307; Sau and Chaput, 2017, Org. Lett.19:4379-4382) using an engineered TNA polymerase that was previouslydeveloped to synthesize TNA on DNA templates (Larsen et al., 2016, Nat.Commun. 7:11235; Chim et al., 2017, Nat Commun 8:1810). The product ofthe primer-extension step is a chimeric TNA-DNA hairpin duplex in whicha 40 nt random region and downstream fixed-sequence primer binding siteare successfully copied into TNA. The TNA portion of the duplex wasdisplaced in a separate step by extending a DNA primer annealed to theloop region of the hairpin with DNA, which results in a combinatoriallibrary of single-stranded TNA molecules that are each physically linkedto their encoding dsDNA templates. To enrich for TNA molecules withaffinity to a specific target, the pool of TNA-dsDNA fusion moleculeswas incubated with a protein target, and bound sequences are recoveredand amplified by PCR. A second PCR step was performed with aPEG-modified DNA primer and the single-stranded, PEGylated DNA templatewas obtained after purification by denaturing polyacrylamide gelelectrophoresis (PAGE). The template strand was then ligated to the DNAstem-loop structure, extended with TNA, and strand displaced toreconstruct the TNA library for another round of in vitro selection.

The DNA display approach was applied to select TNA aptamers withaffinity to a recombinant reverse transcriptase (RT) isolated from thehuman immunodeficiency virus (HIV). HIV RT is the replicative polymerasefor HIV and thus a major target for drug development (Bala et al., 2018,RNA Biol 15:327-337). Although previous selections have generated DNAand FANA (2′-fluoroarabino nucleic acid) aptamers to HIV RT (Michalowskiet al., 2008, Nucleic Acids Res. 36:7124-7135; Alves Ferreira-Bravo,2015, Nucleic Acids Res. 43:9587-9599), both classes of affinityreagents are susceptible to nuclease digestion (Watts et al., 2009, OrgBiomol Chem 7:1904-1910). These studies were extended by establishing abiologically stable aptamer to this target that could function with highbinding affinity under harsh biological conditions, without the need forextensive chemical modifications that have been previously used toimprove the biological stability of DNA aptamers (Eaton et al., 1997,Bioorg. Med. Chem. 5:1087-1096). A key question to address was whetherTNA could fold into structures with the same level of high affinitybinding (picomolar K_(D) values) commonly observed for high qualitymonoclonal antibodies (mAbs). Although a TNA aptamer that binds to HIVRT with a K_(D) of ˜5 nM was previously isolated, that example utilizeda reverse transcription step and required next-generation sequencingdata to identify an aptamer with higher affinity binding (Mei et al.,2018, J. Am. Chem. Soc. 140:5706-5713). Recognizing that the current TNAreverse transcriptase functions with weak activity (Dunn and Chaput,2016, ChemBioChem 17:1804-1808), without being bound by theory, it wasreasoned that a DNA display approach should make it possible to recoverhigh affinity sequences that may have been lost in the previousselection due to the inherent limitations of the current TNA reversetranscriptase (Mei et al., 2018, J. Am. Chem. Soc. 140:5706-5713).

Three rounds of selection were performed starting from a population of10¹³ different TNA molecules, each displayed on their encoding dsDNA.For each round of selection, the library was incubated with HIV RT,which was immobilized on the surface of a functionalized and passivatedwell of an amino modified-ELISA plate. After a 1-hour incubation (25°C.), the well was drained, unbound sequences were removed by washingwith buffer, and non-specific interactions were disrupted throughadditional washing steps with a high ionic strength buffer. Highaffinity aptamers that remained bound to the target were then recoveredby denaturing the complex at 70° C. with buffer containing 3.4 M urea.After three rounds of selective amplification, a portion of the pool wascloned into E. coli and 20 library members were submitted for Sangersequencing. From this set, 15 high quality reads were obtained with nosignificant sequence similarity. Nine randomly selected clones wereexperimentally synthesized by primer-extension and their dissociationconstants (K_(D)) were determined under equilibrium conditions bymicroscale thermophoresis (MST). The screen for protein binding activityresulted in K_(D) values ranging from 400 pM to 70 nM with six of thenine sequences having K_(D) values of <10 nM. The three highest affinityTNA aptamers (HIV-RT 3.10, HIV-RT 3.15, and HIV-RT 3.17) bound to HIV RTwith K_(DS) of 4 nM, 2 nM, and 380 pM, respectively (FIG. 4C). Thehighest affinity aptamer, HIV-RT 3.17 (K_(D) 380±115 pM), was evaluatedin triplicate to ensure the reproducibility of our measurements.

To validate the affinity of HIV-RT 3.17, a second analytical techniquewas used to measure the K_(D) of the aptamer to HIV RT. In this case, anelectrophoretic mobility shift assay (EMSA) was used to evaluate thebinding interaction by incubating the protein with a low concentrationof labeled aptamer and varying the concentration of the protein target(FIG. 5A). As a positive control, the K_(D) of R1T, a known DNA aptamerpreviously selected by Burke and colleagues to bind HIV RT (Michalowskiet al., 2008, Nucleic Acids Res. 36:7124-7135), was measured. Theresulting isotherms reveal that HIV-RT 3.17 binds to HIV RT with a K_(D)of 196±86 pM and R1T binds with a K_(D) of 4.5±0.41 nM. These valuesclosely agree with the MST derived value of 380 pM for HIV-RT 3.17 andthe literature K_(D) for R1T (14±2 nM).

Next, the binding properties of HIV-RT 3.17 were evaluated underconditions where DNA aptamers and monoclonal antibodies are known tolose their activity. Since most aptamers are susceptible to nucleasedigestion, the HIV RT binding assay was performed in the presence ofsnake venom phosphodiesterase (SVPE), a highly active 3′ exonucleasethat is used to degrade DNA and RNA into mononucleotides (Bowman et al.,2001, Nucleic Acids Res. 29:E101). In this assay, the HIV-RT 3.17 andR1T aptamers were incubated with SVPE for 1 hour at 37° C. prior toperforming the equilibrium binding assay by EMSA. Analysis of theresulting binding curves revealed that HIV-RT 3.17 remains active in thepresence of SVPE (K_(D) of ˜500 pM), while DNA digestion abrogates allprotein-binding activity for the R1T aptamer (FIG. 5D). Time-dependentanalysis of R1T digestion by denaturing PAGE reveals that the DNAaptamer degrades in minutes, while the TNA aptamer remains undigestedafter 24 hours of incubation at 37° C. (FIG. 5C).

Recognizing that antibodies are prone to rapid and irreversibleunfolding at ambient temperature, a thermal challenge was performed byheating HIV-RT 3.17, R1T, and a commercial monoclonal antibody generatedagainst HIV RT in buffer at 70° C. At designated time points between 0and 48 hours, the reagents were cooled to room temperature and assayedfor binding to HIV RT by biolayer interferometry (BLI). The Kis fromthis data were used to calculate changes in the Gibbs free energy atstandard conditions (ΔG°), which were plotted as a function oftemperature to illustrate the effect of heating on the affinity reagents(FIG. 5D). Visualizing these data in terms of binding energy as opposedto affinity affords a more intuitive picture of the effect of heating onthe function of each binder. The results indicate that HIV-RT 3.17 andR1T retain full activity after 48 hours of heating at 70° C., while themonoclonal antibody lost activity within the first hour of heating.After 72 hours, the activities of both aptamers are reduced to 50%,presumably due to problems associated with thermal degradation.

To gain further insight into the function of HIV-RT 3.17, a secondarystructure prediction calculation was performed using mFold (Zuker, 2003,Nucleic Acids Res. 31:3406-3415). Although mFold was developed topredict the secondary structure of DNA and RNA oligonucleotides, thealgorithm favors the formation of Watson-Crick base pairs, making it areasonable model for unnatural genetic polymers with phosphodiesterbackbones. HIV-RT 3.17 was predicted to adopt a step loop structure thatcontains two bulges (one large and one small) in the stem region (FIG.4C). It also contains a predicted T:G wobble base pair and flankingsequences on both the 5′ and 3′ sides of the primary stem loopstructure. Detailed structure-activity relationship studies confirmedthe importance of both the stem loop structure and the wobble base pairfor the bind to HIV RT. In general, deletions made throughout theprimary stem loop structure of the HIV-RT 3.17 molecule reduced thebinding activity by 2-10-fold. However, more significant drops inactivity (17-100-fold) were observed when changes were made to the fivebase pairs that define the stem near the 5′ and 3′ termini. Even themutation of a T:G wobble pair to a standard Watson-Crick C:G baseresulted in a 10-fold loss in activity. Deletion of either the two 5′flanking nucleotides or the five 3′ flanking nucleotides resulted in a˜3-fold reduction in binding affinity.

Confident that HIV-RT 3.17 adopts a stem-loop structure, twosplit-aptamers were designed that function with Kis of 40-50 nM (FIG.6). The split-aptamer design is a common approach for biological sensorsand has not previously been demonstrated for XNA polymers.Interestingly, replacing the five base pair stem in the primarystem-loop structure in both the unsplit and split aptamers resulted in asimilar reduction in binding affinity. This suggests that the precisestem structure selected for in HIV-RT 3.17 likely provides more thanstructural integrity and may manifest in a more complex level ofinterection with its protein target. More engineering of the splitaptamer system based on the HIV1-RT structure may yield a split aptamersystem with low nanomolar to picomolar affinity.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of producing a monoclonal xeno-nucleicacid aptamer particle (mXNAP) display library comprising: (a) providinga population of particles comprising a plurality of clonal template DNAmolecules, each of which comprises a DNA coding region and a fixedsequence primer binding site; (b) ligating a self-priming stem-loopforming hairpin DNA molecule to the 3′ end of the template DNAmolecules; (c) extending the 3′ end of the self-priming stem-loop in thepresence of a polymerase capable of synthesizing an XNA from a DNAtemplate and one or more XNA triphosphate molecules (xNTPs) to form apopulation of particles comprising clonal double stranded XNA-DNA hybridmolecules; (d) contacting the double stranded XNA-DNA display templateswith a primer which anneals to the loop region of the stem-loopstructure; and (e) extending the DNA primer using dNTPs and a DNApolymerase to displace the XNA portion of the XNA-DNA display templatesto form a population of mXNAP display particles comprising a pluralityof clonal nucleic acid molecules comprising a dsDNA region and asingle-stranded XNA region.
 2. The method of claim 1, wherein the mXNAPlibrary is selected from the group consisting of a mTNAP, mHNAP, mCeNAP,mLNAP, mANAP, mphNAP, and mFANAP library.
 3. The method of claim 2,wherein the polymerase of (c) is selected from the group consisting ofKod-RSGA, Kod-RS, Kod-RI, Therminator, pol6G12_521L, pol6G12, polC7,polD4K, PGV2, D4K enzyme, 9oN DNA polymerase, Tgo DNA polymerase, KodDNA polymerase and Deep vent DNA polymerase.
 4. A mXNAP display librarycomprising a population of display particles, wherein each displayparticle comprises a plurality of clonal nucleic acid moleculescomprising a dsDNA region and a single-stranded XNA aptamer region. 5.The mXNAP display library of claim 4, wherein the mXNAP library isselected from the group consisting of a mTNAP, mHNAP, mCeNAP, mLNAP,mANAP, mphNAP, and mFANAP display library.
 6. The mXNAP display libraryof claim 4 comprising at least 10⁵ mXNAPs.
 7. A method of screening foran XNA aptamer having a desired property, comprising the steps of: (a)incubating a mXNAP display library of claim 4 with at least onecandidate interaction partner for an amount of time sufficient forinteraction of the XNA aptamer regions with the candidate interactionpartner; and (b) selecting mXNAP particles displaying XNA aptamers thathave the desired property.
 8. The method of claim 7, wherein the desiredproperty is selected from the group consisting of a target bindingactivity and a target-binding induced activity.
 9. An XNA aptameridentified according to the method of claim 7 as having a desiredproperty.
 10. The XNA aptamer of claim 9, wherein the aptamer isselected from the group consisting of a TNA aptamer, a HNA aptamer, aCeNA aptamer, a LNA aptamer, an ANA aptamer, a phNA aptamer and a FANAaptamer.
 11. A pharmaceutical composition comprising an XNA aptamer ofclaim
 9. 12. The pharmaceutical composition of claim 11, furthercomprising a pharmaceutically acceptable excipient.
 13. A method oftreating a disease or disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition of claim
 10. 14. A TNA aptamer, comprising a sequenceselected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3, avariant of SEQ ID NO:1 to SEQ ID NO:3, and a fragment comprising atleast 20 nt of SEQ ID NO:1 to SEQ ID NO:3.
 15. A pharmaceuticalcomposition comprising the TNA aptamer of claim
 14. 16. Thepharmaceutical composition of claim 15, further comprising apharmaceutically acceptable excipient.
 17. A method of treating HIV or adisease or disorder associated therewith in a subject in need thereof,comprising administering to the subject an effective amount of thepharmaceutical composition of claim 15.